Development of masked therapeutic antibodies to limit off-target effects

ABSTRACT

In one embodiment, a masked monoclonal antibody (mAb) is provided, the mAb, encoded by a nucleic acid sequence or an amino acid sequence molecule comprising a signal sequence, a masking epitope sequence, a linker sequence that is cleavable by a protease specific to a target tissue; and an antibody or a functional fragment thereof. In another embodiment, a masked monoclonal antibody (mAb) is provided, which includes a therapeutic mAb and a mask, the mask comprising protein A and protein L attached by a protease cleavable linker.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.13/424,272, filed Mar. 19, 2012, issued as U.S. Pat. No. 9,193,791,issued Nov. 24, 2015, which claims priority to U.S. Provisional PatentApplication No. 61/454,500, filed Mar. 19, 2011; and is acontinuation-in-part of U.S. application Ser. No. 12/849,786, filed Aug.3, 2010, all of which are hereby incorporated by reference as if fullyset forth herein.

STATEMENT OF GOVERNMENT SUPPORT

The government has certain rights in the present invention. The presentinvention was made with government support under National Institutes ofHealth Grant Nos. R21CA135216 and S10 RR022316; and Grant No.W911QY-10-C-0176, awarded by the Department of Defense (DOD). TheGovernment has certain rights in the invention.

BACKGROUND

Current monoclonal therapies for both leukemia and solid tumors sufferfrom a lack of specificity. Most often the targeted epitopes are nottumor specific, but are also present in other non-diseased tissues. Inthe case of Cetuximab and Matuzumab, expression of EGFR in hairfollicles, the intestines and kidney leads to non-tumor toxicity.Patients experience an acneaform rash, gastrointestinal toxicity andhypomagnesemia that may limit the duration of therapy. For Trastuzumab,cardiotoxicity is observed because of the role that ErbB2 plays incardiomyocyte health. The antibody exacerbates the cardiotoxicity ofanthracyclines necessitating years of surveillance for development ofdilated cardiomyopathy. For these antibodies and others it would bebeneficial to improve tumor selectivity.

In addition to off-target effects, antibody therapies against solidtumors face other challenges. First, tumor vasculature is leaky,resulting in high interstitial pressures that any molecule entering thetumor has to overcome. Second, high affinity antibodies are needed tostay in the tumor long enough to exert their effects, but high affinityantibodies may encounter a “binding site barrier” where they weretrapped by the peripheral antigen and never diffuse into the center of asolid tumor. This may result in underexposure of the tumor center. Itwould therefore be desired to develop antibodies and methods for theirdelivery to tumor cells that minimize effects to non-diseased tissues.

SUMMARY

Therapeutic antibodies cause side effects by binding receptors innon-target tissues. In one embodiment, a “prodrug” antibody design isdescribed that ameliorates such side effects by occluding (directly orsterically), or “masking” antibody complement determining regions untilthey reach diseased tissues containing disease-associated proteinases.In one embodiment, a masked mAb may comprise a nucleotide sequence whichencodes a first segment comprising a signal sequence; a second segmentcomprising a masking epitope sequence, wherein the masking epitopesequence contains an epitope specific to the mAb; a third segmentcomprising a cleavable linker sequence; and a fourth segment comprisingan antibody or functional fragment thereof. In some embodiments, thefourth segment is a single chain variable fragment (scFv). In otherembodiments, the fourth segment may be an IgG.

In some embodiments, two mAbs may form a heterodimer to produce across-masked mAb complex, comprising a first masked mAb comprising (1) afirst masked antibody or fragment thereof having an antigen recognitionsite attached to a first masking epitope via a flexible linker, and (2)a second masked mAb comprising a second antibody or fragment thereofhaving an antigen recognition site attached to a second masking epitopevia a flexible linker. The first and second masked mAbs may form aheterodimer complex by occlusion of the first and second antigenrecognition sites by the second and first masking epitopes,respectively. The flexible linker may be cleaved by a protease specificto a target tissue allowing the cross-masked mAb heterodimer complex todissociate at the target tissue. In some aspects, masked mAbs againstthe epidermal growth factor receptor (EGFR) were fused with a linkerthat is susceptible to cleavage by a proteinase to their epitope.

In another embodiment, a masked mAb may include a mAb bound by a proteinA-protein L mask (protein A-L) mask. In another embodiment, protein Aand/or protein L may be used as crystallization additives for a Fab.

Surface plasmon resonance and flow cytometry were used to confirm thatbinding is dependent on proteinase release. These molecular designs aregenerally applicable to other therapeutic antibodies to increase theirspecificity for diseased tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C illustrate the combinatorial effects of mAbs 425 and C225 onproliferation and anchorage-independent cell survival of MDA-MB-468breast cancer cells. FIG. 1A shows dose-dependent effects of eitherantibody alone at 10 μg/ml and the antibody combination at 10 μg/ml onmetabolic activity as determined by WST-1 assay; the experiment wasperformed three times and results shown as m±SEM. FIG. 1B showsisobolographic representation of synergistic antibody effects at 25%growth inhibition. FIG. 1C shows impaired survival of MDA-MB-468 cellsin forced suspension culture in the presence of the 425/C225combination. In these experiments, the EGFR selective kinase inhibitor,AG1478, was used as a positive control. The capacity of cells toreattach and resume proliferation after two days of forced suspensionculture in the presence or absence of 10% FCS was determined. Reattachedcells were visualized by crystal violet staining 24 h after reseeding oncell culture-treated plastic

FIG. 2 illustrates the effects of mAbs 425 and C225 alone and incombination on signal transduction events upon EGF treatment ofMDA-MB-468 cells. Phosphorylation of signaling intermediaries(p-AKTS473; p-42/44MAPK; p-EGFRY1068) was determined by immunoblotanalysis using phosphospecific antibodies for up to one hour afteraddition of EGF (10 ng/ml) to cells. Comparable loading of wells wasassessed using antibodies recognizing the EGFR and β-actin,respectively. Use of the antibody combination amplified inhibitoryeffects of C225 on AKT and MAPK phosphorylation. Representative resultsof experiments performed three times are shown.

FIGS. 3A-C illustrate simultaneous binding of mAbs C225 and 425 to theextracellular portion of the EGFR (sEGFR). FIG. 3A shows surface plasmonresonance analysis of sEGFR captured by 425 tethered on CM5 chips. Thereal time sensorgram for each different sEGFR concentration (lighter,wavy lines) is superimposed with the calculated fit using the model of1:1 Langmuir binding with mass transport limitation (fitted smooth blacklines). The residuals of the fit are provided under the sensorgram. Forcalculation of binding affinities please refer to Table 1. FIG. 3B showssurface plasmon resonance analysis of sEGFR captured by C225 bound toCM5 chips. FIG. 3C shows binding of 425 to sEGFR captured by C225.Approximately 30 RUs of sEGFR were captured on a C225 immobilized CM5chip and used as ligand to study the binding kinetics of mAb 425.Increasing concentrations of 425 were injected at 20 μl/min for twominutes association time and two minutes dissociation time. All Biacoreexperiments shown were conducted at least three times with similarresults.

FIGS. 4A-B illustrate sedimentation equilibrium analysis of complexesformed by C225 and 425 Fabs and the extracellular portion of EGFR(sEGFR). FIG. 4A shows that the complex was formed by saturating sEGFRwith Fab fragments of C225 and 425 and isolated by size exclusionchromatography. FIG. 4B shows radial scans at 280 nm were collected at8000, 12000 and 16000 RPM at 20° C. The data fit well to a singlemolecular species and afforded a calculated molecular weight of167,100+/−1000 Da, consistent with a tripartite complex.

FIGS. 5A-D illustrate independent binding of mAbs C225 and 425 to thehuman EGFR expressed on cell surfaces. Binding of Alexa Fluor488-labeled C225 and 425 was assessed by FACS in the presence ofunlabeled C225 or 425 as indicated in the panels. This analysis wasperformed using NIH3T3 cells engineered to express wild-type human EGFR(HC2; FIGS. 5A and 5B) or the tumor-specific EGFRvIII (C012; FIGS. 5Cand 5D) as indicated. In both cases, either antibody competed withitself but not with the other antibody. A representative example ofthree experiments is shown.

FIG. 6 illustrates modeling of the 425 binding site on the EGFR. Surfacerepresentation of the extracellular portion of EGFR bound to C225(ribbon representation) based on the structure 1YY9 (Li et al., 2005).Glycosylation of asparagine residues found in the structure of 1YY9 areshown as sticks. The EGF-EGFR interface based on the crystal structure1IVO (Ogiso et al., 2002) and limited to 5 Å cutoff is shown in thefigure as “EGF Interface”. Note that S460 and G461 represent the onlysurface residues of interest on domain III that are either not occludedby C225 or likely to be affected by N-linked glycosylation. The figurewas made in PyMol (DeLano, 2002).

FIG. 7 illustrates that MAb 425 binds the EGFR at an epitope distinctfrom C225 as determined by size exclusion chromatography. mAbs 425 andC225 bind to domain III individually (Line 4 and Line 8) and as acombination (Line 9). Note that the complex of 425 with theEGFRdomIIIS460P/G461N elutes slightly earlier than the individualcomponents (Line 5), but significantly later than the non-mutated domainIII (Line 4). The complex of C225 with the mutated EGFRdIII (Line 7)eluted at the same volume as the non-mutated domain III (Line 8)indicating that the point mutations do not interfere with the overalltertiary structure. Asterisks denote an impurity present in the C225preparation. The concentration of each sample added to the column was 4μM (based on absorbance at 280 nM).

FIGS. 8A-B are schematic views illustrating the masked antibody (or“prodrug”) concept in accordance with one embodiment. FIG. 8Aillustrates the proof-of-principle, and FIG. 8B is a schematic view ofthe overall design to generate IgGs that are masked and do not bindantigens in normal tissues.

FIGS. 9A-C illustrate the design, production and characterization ofcross-masked 425/C225 scFvs in accordance with one embodiment. FIG. 9Ais a schematic diagram illustrating the topology of masked scFvconstructs indicating point mutations in EGFRdIII for either mask. FIG.9B is a graph illustrating the individual masked scFvs are monomericwhereas admixture of C225 and 425 cross-masked scFv is consistent with aheterodimeric complex as illustrated by size exclusion chromatography inaccordance with one embodiment. FIG. 9C illustrates specific cleavage ofcrossmasked heterodimeric scFvs and individual masked scFvs by MMP9 asdetermined by SDS-PAGE.

FIGS. 10A-B illustrate the binding analysis of cross-masked 425/C225scFvs in accordance with one embodiment. FIG. 10A shows surface plasmonresonance analysis of cross-masked 425/C225 binding at 1 μM and 100 nMto immobilized sEGFRdIII before and after MMP-9 digestion (indicated byarrow). FIG. 10B shows FACS analysis of scFv binding reveals that afterMMP-9 digestion the cross-masked 425/C225 binds to HaCaT cells as wellas the native scFvs. IgG 425 binding confirms EGFR expression.

FIG. 11 is an elution profile from Superdex 200 10/300 size exclusioncolumn of masked (native domain III mask) C225 before and after MMP-9treatment in accordance with one embodiment. Earlier elution in theabsence of MMP-9 indicates the presence of a homodimeric cross-maskedspecies that resolves into two non-covalent scFv-mask complexes. Elutionvolumes from the same column of the singly masked scFvs (with pointmutated domain III masks) and cross-masked C225/425 are shown forcomparison.

FIGS. 12A-B are graphical representations of traces from surface plasmonresonance affinity determinations for the singly masked C225 and 425 inaccordance with one embodiment. The affinities are nearly identicalbefore and after MMP-9 digestion. This suggests that the point mutationsreduce the apparent affinity of the homodimer resulting in no masking.

FIG. 13 is a flow cytometry graph illustrating that masked 425 alonedoes not exhibit an MMP-9 dependent increase in binding. However, thesEGFRdIII-antibody fusion is free to bind before linker digestion,indicating a lack of masking.

FIG. 14 shows the nucleotide (SEQ ID NO:1) and amino acid (SEQ ID NO:5)sequences of masked C225 (Signalsequence-sEGFRdIII(Q384A/Q408M/H409E)-Linker(MMP-9 site)-scFv C225(V_(L)-linker-V_(H))-H₆).

FIG. 15 shows the nucleotide (SEQ ID NO:2) and amino acid (SEQ ID NO:6)sequences of masked 425 (Signalsequence-sEGFRdIII(S460P/G461N)-Linker(MMP-9 site)-scFv 425(V_(L)-linker-V_(H))-FLAG tag-H₆).

FIGS. 16A-B show a proposed molecular design to increase tumor selectionand reduce off-target effects of therapeutic antibodies. FIG. 16A showsthat by targeting cells that have increased antigen expression, whichresults in targeting of normal tissues with high antigen expression.FIG. 16B shows that increased specificity may be attained by designingantibodies that are activated selectively by proteases within tumortissue (e.g., MMP-9).

FIG. 17 shows the masked antibody C225 and 425 constructs. Antibodieswere assembled to include a “mask” comprised of domain III of the EGFRand a linker containing an MMP-9 consensus protease site. Pointmutations corresponding to the epitope of the attached antibody wereintroduced into the domain III masks. The native EGFR signal sequencewas used for secretion. Both antibodies were scFv constructs assembledwith the light chain into the heavy chain. Only masked 425 contained aFLAG tag, while both antibodies contained a hexa-histadine tag thatadded in purification.

FIG. 18 shows a schematic diagram of a mask design for Cetuximabaccording to one embodiment. EGFR domain III is linked to the heavychain of Cetuximab via an MMP9 cleavable (top line) or non-cleavable(bottom line) linker (left panel). The right panel shows the mask-heavychain construct (dill Linker HC) and the light chain (LC) of Cetuximabcloned into a dual expression GS selection vector.

FIG. 19 is a Western blot that illustrates transient and stableexpression of masked Cetuximab with an anti-human Fc-HRP antibody ofCOS-7 culture supernatants three days after transient transfection (leftpanel); and a dot blot identification of stably-transfectedmasked-Cetuximab NS0 cell lines. Culture supernatant of single colonyclones were spotted and blotted by anti-human Fc-HRP antibody.

FIG. 20 shows a non-reducing (left) and reducing (right) SDS-PAGE ofmasked Cetuximab with cleavable (WMC) and non-cleavable (XMC) linker.Expected molecular weight (MW) of masked Cetuximab (MC)=200 kD,mask-HC=75 kD, LC=24 kD.

FIG. 21 shows a reducing SDS-PAGE of MMP9 treated with masked Cetuximabwith cleavable (WMC) and non-cleavable (XMC) linker.

FIGS. 22A-E illustrate sedimentation equilibrium analysis of (FIG. 22A)masked Cetuximab with a cleavable linker (WMC), (FIG. 22B) maskedCetuximab with a non-cleavable (XMC) linker, (FIG. 22C) WMC-Matuzumabcross-masked complex (WMC+M425), (FIG. 22D) XMC-Matuzumab cross-maskedcomplex (XMC+M425), and (FIG. 22E) Matuzumab alone (M425). The dataafforded a calculated molecular weight of 145,180 (M425), 219,890 (WMC),226,940 (XMC), 239,320 (WMC+M425) and 353,190 (XMC+M425).

FIG. 23 is a size-exclusion chromatography (SEC) elution analysis of theinteraction of masked Cetuximab with a non-cleavable linker (XMC) andMatuzumab (M425). Peak fractions from XMC+M425, XMC and M425 wereresolved on a non-reducing SDS-PAGE (left inset).

FIG. 24 is a size-exclusion chromatography (SEC) elution analysis of theinteraction of masked Cetuximab with a cleavable linker (WMC) andMatuzumab (M425).

FIG. 25 shows the results of a FACS analysis of maskedCetuximab-matuzamb complex binding to MDA-MB-468 cells. Alexa647-labeled XMC complexed with M425 were isolated from SEC, treated withMMP9, and mixed with trypsinized MDA-MB-468 cells, M425 were labeledwith Alexa 488 anti-human IgG secondary antibodies.

FIG. 26 shows the results of a FACS analysis of maskedCetuximab-matuzamb complex binding to MDA-MB-468 cells. Alexa647-labeled WMC complexed with M425 were isolated from SEC, treated withMMP9, and mixed with trypsinized MDA-MB-468 cells, M425 were labeledwith Alexa 488 anti-human IgG secondary antibodies.

FIG. 27 shows a schematic diagram of a mask design for Trastuzumabaccording to one embodiment. HER2 domain IV is linked to the Fc regionof Trastuzumab via an MMP9 cleavable (top line; SEQ ID NO:7) ornon-cleavable (bottom line; SEQ ID NO:8) linker.

FIG. 28 is a non-reducing SDS-PAGE of the supernatant (sup.), flowthrough (FT) and the eluted protein (Elu.) from culture of baculovirusesthat were transfected with the mask-Trastuzumab complex described inFIG. 27. The expected MW of the H4Fc dimer is 79 kDa, which is the bandmarked by (*).

FIG. 29 is a portion of a size exclusion chromatography (SEC) trace(bottom) was shown on the right, and a non-reducing SDS-PAGE (top)showing the H4Fc eluted from fractions 22-24 (F20-F24), as well as twoaggregation species in F17 and F20 and a degradation product, likely theFc portion, in F26.

FIG. 30 shows surface plasmon resonance analyses of H4Fc including acleavable linker (HW; top trace) or of H4Fc that includes anon-cleavable linker (HX; bottom trace) captured by the Fab portion ofTrastuzumab tethered on CM5 chips.

FIG. 31 is a non-reducing SDS-PAGE to resolve various proteincombinations (Trastuzumab, HW, HX, MMP9) as indicated.

FIG. 32 shows results of a FACS analysis of Alexa 488 labeledTrastuzumab incubated with either H4Fc's at a 1:1 or 1:2 ratio for 20min at room temperature, with or without MMP9 as indicated.

FIG. 33 illustrates the masking scheme for a generic mask to amonoclonal antibody. Upon interaction with a tumor-specific protease (b)(e.g., MMP9 or ADAM10), a masked monoclonal antibody (a), releases theprotein A and L mask (c, d) and is free to bind target receptors ontumor cells.

FIGS. 34A-D show the interaction between protein A and Protein L withthe heavy chain and light chain of the Trastuzumab Fab (FIGS. 34A, 34B)and the crystal structures of Proteins A and L bound to the TrastuzumabFab (FIGS. 34C, 34D).

FIG. 35 is a table showing the X-ray diffraction data for TrastuzumabFab.

FIGS. 36A-D illustrate sedimentation equilibrium analysis of a proteinA-protein L mask, a Trastuzumab Fab, and a protein A-protein Lmask/Trastuzumab Fab complex. FIG. 36A shows isolation of the proteinA-protein L mask (mask), the Trastuzumab Fab (Fab), and a proteinA-protein L mask:Trastuzumab Fab complex (Trastuzumab Fab+mask) by sizeexclusion chromatography. FIGS. 36B-D show radial scans at 280 nm. Thedata afforded a calculated molecular weight of 162,082+/−162.70 (mask),45,909+/−166.41 (Fab) and 56,380+/−160.33 (mask+Fab), consistent with a1:1 complex.

FIGS. 37A-C show surface plasmon resonance (SPR) analyses of the bindingaffinity of protein A (FIG. 37a ), protein L (FIG. 37b ) and proteinA-protein L mask for Trastuzumab Fab (FIG. 37c ), The concentrationrange for a-b is 3.2 nM to 5 μM and 10 pM to 1 μM.

FIGS. 38A-D show a comparison of on-rates of masked and unmaskedTrastuzumab Fab binding to HER2 by illustrating an SPR Analysis ofTrastuzumab Fab binding to HER2 (FIGS. 38a-38b ) and Trastuzumab Fabwith excess protein A-protein L mask binding to HER2 (FIGS. 38c-38d ).Panels b and d compare the on rates of the two Fab to HER2.

DETAILED DESCRIPTION

The following description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure.

Monoclonal antibodies are increasingly being used in the clinicalmanagement of diverse disease states, including cancer (Adams andWeiner, 2005). Monoclonal antibodies (mAbs) that inhibit activation ofthe epidermal growth factor receptor (EGFR) have shown therapeuticpotential in select malignancies including breast cancer. As describedin Example 1 below, the combined use of two such mAbs, C225 (Cetuximab)and 425 (EMD55900), reduces growth and survival of EGFR overexpressingMDA-MB-468 breast cancer cells more effectively than either antibodyalone. Similarly, the C225/425 antibody combination more effectivelyinhibited AKT and MAPK phosphorylation in MDA-MB-468 cells. Surfaceplasmon resonance, size exclusion chromatography and analyticalultracentrifugation demonstrated that mAbs C225 and 425 simultaneouslybind to distinct antigenic epitopes on domain III of the solublewild-type EGFR. Furthermore, neither mAb competed with the other forbinding to cells expressing either wild-type EGFR or a mutant EGFR(EGFRvIII) associated with neoplasia. Mutagenesis experiments revealedthat residues S460/G461 in EGFR domain III are essential components ofthe 425 epitope and clearly distinguish it from the EGF/TGFα bindingsite and the C225 interaction interface. Collectively, these resultssupport the conclusion that therapeutic EGFR blockade in cancer patientsby combined use of mAbs C225 and 425 could provide advantages over theuse of the two antibodies as single agents.

When used as ‘targeted agents,’ mAbs generally cause fewer severe sideeffects than traditional chemotherapy. However, adverse events have beenreported and described for many antibody therapeutics due to inadvertentantigen recognition in normal tissues. In the case of epidermal growthfactor receptor (EGFR) antagonistic mAbs, dose-limiting toxicities arethought to be due to engagement of the receptor by the therapeuticantibody in normal tissues (Lacouture et al., 2006; Rodeck, 2009).

The Erb tyrosine kinase family includes four members, of which the EGFRand Her2 are frequently deregulated in solid tumors and are ofsignificant interest as therapeutic targets. MAbs to both antigens areused to treat various epithelial cancers. However, EGFR antagonisticmAbs, including Cetuximab (Masui et al., 1984), Matuzumab (Rodeck etal., 1987), and the fully human Panitumumab (Segaert and Van Cutsem,2005; Van Cutsem et al., 2008), can cause dose-limiting adverse eventsaffecting primarily the skin and the gastrointestinal system (Vanhoeferet al., 2004).

To address undesirable side effects (or “off-target” effects) caused bymAbs, antibody “prodrugs” were developed and tested as described herein.The antibody prodrug design is based on non-covalent proteininteractions with the unmodified antibody to occlude of the antigenrecognition sites (e.g., the complement determination regions (CDRs)) ofmAbs or modulate the dynamics of the CDR through fusion with recombinantantigen fragments (also known as “masking epitopes”) via a flexiblelinker. Invasive tumors typically express proteolytic enzymes, such asmatrix metalloproteinases (MMPs), to breakdown the extracellular matrixfor invasion and metastasis. Although the MMP family has at least 28members, MMP-9 is known to correlate with malignancies that respond toepidermal growth factor blockade (Zhou et al. 2006; Swinson et al. 2004;Cox et al. 2000). The presence of these enzymes can distinguish tumortissue from normal tissue.

Occluded antibody prodrugs that are described herein are also known as‘masked’ antibodies and their activated counterparts are also known as‘unmasked’ antibodies. Occluded or masked mAbs are prevented frombinding in normal tissues that express the epitope but do not expressMMPs. However, the mask is designed to be susceptible to MMPs. Masked oroccluded mAbs may be ‘activated’ by including tumor protease specificsequences in the linker so that digestion by MMPs causes the mask tofall off. This adds an additional dimension to the current principle oftumor selectivity. Cell surface receptor (epitope) density is acceptedas a primary selection basis for antibody targeting. Since thesereceptors exist in normal tissues, the antibodies also exert theireffects in healthy tissues (FIG. 16A). By adding an additional selectionproperty (i.e., MMP-9 expression), increased tumor specificity may beobtained. (FIG. 16B) Masked and unmasked antibody design has been testedusing two EGFR antagonistic antibodies, 425 (Matuzumab) and C225(Cetuximab); and a HER2 antibody, Trastuzumab.

Any mAb may be developed as a masked antibody. In some embodiments, themasked antibody may be a masked anti-EGFR. The epidermal growth factorreceptor (EGFR; ErbB-1; HER1) is one of four members of the ErbBreceptor family and contributes to growth, survival, migration anddifferentiation of epithelial cells (Yarden and Sliwkowski, 2001).Deregulated signaling through the EGFR either alone or in cooperationwith other members of the ErbB family, notably ErbB2 and ErbB3, is ahallmark of multiple neoplasms predominantly of epithelial origin. Themolecular mechanisms leading to deregulated EGFR-dependent signalinginclude overexpression of the EGFR, establishment of autocrine loops byaberrant overexpression of EGFR ligands and, the expression of mutated,constitutively active EGFRs (Mendelsohn and Baselga, 2000; Kim et al.,2001; Nagane et al., 2001).

In recognition of the potential roles of aberrant EGFR activation intumor progression, multiple antagonists of EGFR activation have beendeveloped with therapeutic intent. These can be broadly divided into twoclasses: (i) small molecules that target the kinase domain of the EGFRand inhibit its phosphorylation activity and (ii) monoclonal antibodies(mAbs) binding to the extracellular domain of the EGFR (Baselga andArteaga, 2005). Typically, EGFR antagonistic mAbs were selected todisrupt ligand binding to the extracellular domain of the wild-typeEGFR. Two examples of EGFR mAbs are the murine mAb 225 and the murinemAb 425. A chimeric version of 225 (C225; Cetuximab; Erbitux) containinga human Fc fragment has been FDA-approved for treatment of severalepithelial neoplasias including colorectal carcinoma. A humanizedversion of 425 (EMD72000; Matuzumab) is currently in Phase II clinicaltrials in various epithelial neoplasms.

In other embodiments, the masked antibody may be a masked anti-HER2.HER2/ErBB2/Neu is a tyrosine kinase protein receptor which is expressedin the heart, lung, intestine and kidney and its activation is involvedin growth and proliferation. HER2 is up-regulated in an aggressive formof metastatic breast cancer making it a potential target for cancertherapy. Herceptin®/Trastuzumab is a therapeutic humanized monoclonalantibody used to treat HER2 positive breast cancer.

Trastuzumab has been shown to elicit adverse side effects which arethought to be caused by off target binding of the antibody to HER2expressed in healthy tissues at normal levels. This problem is notlimited to Trastuzumab or to mAbs in general, in fact most targetedtherapies target a mechanism rather than or in addition to the diseasesite. These adverse side effects could be prevented by specificallytargeting the tumor cells rather than other cells that express HER2 atlower levels.

In some embodiments, a masked antibody may comprise a nucleotidesequence which encodes a first segment comprising a signal sequence; asecond segment comprising a masking epitope sequence, wherein themasking epitope sequence contains an epitope specific to the mAb, forexample the masking epitope sequence is specific to one or more antigenrecognition sites (e.g., CDRs) of the mAb (i.e., is specific to thefourth segment, described below); a third segment comprising a cleavablelinker sequence; and a fourth segment comprising an antibody orfunctional fragment thereof.

An antibody or functional antibody fragment thereof refers to animmunoglobulin (Ig) molecule that specifically binds to, or isimmunologically reactive with a particular antigen, and includes bothpolyclonal and monoclonal antibodies. The term antibody includesgenetically engineered or otherwise modified forms of immunoglobulins,such as intrabodies, chimeric antibodies, fully human antibodies,humanized antibodies, peptibodies and heteroconjugate antibodies (e.g.,bispecific antibodies, diabodies, triabodies, and tetrabodies). The termfunctional antibody fragment includes antigen binding fragments ofantibodies, including e.g., Fab′, F(ab′)₂, Fab, Fv, rIgG, and scFvfragments. The term scFv refers to a single chain Fv antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.

In some embodiments, the fourth segment is a single chain variablefragment (scFv). Examples of individual masked antibodies are shown inFIGS. 14 (masked C225; SEQ ID NO:1 (nucleic acid); SEQ ID NO:5 (aminoacid)) and 15 (masked 425; SEQ ID NO:2 (nucleic acid); SEQ ID NO:6(amino acid)). In some embodiments, the fourth segment may be an IgG.The use of an IgG creates a bivalent format with an enhanced affinity tothe target.

In some embodiments, two mAbs may form a heterodimer to produce across-masked mAb complex, comprising a first masked mAb comprising (1) afirst antibody or fragment thereof having an antigen recognition siteattached to a first masking epitope via a flexible linker, and (2) asecond masked mAb comprising a second antibody or fragment thereofhaving an antigen recognition site attached to a second masking epitopevia a flexible linker. The first and second masked mAbs may form aheterodimer complex by occlusion of the first and second antigenrecognition sites by the first and second masking epitopes. The flexiblelinker may be cleaved by a protease specific to a target tissue allowingthe cross-masked mAb heterodimer complex to dissociate at the targettissue.

In one embodiment of the disclosure, the EGFR antagonistic antibodies,425 and C225 were selected for development of cross-masked antibodies.As shown in FIG. 8A, EGFR domain III, which has an epitope for both 425and C225, was fused via a cleavable linker to an scFv of C225 and of425. Point mutations in EGFR domain III favor a heterodimer. When themasked antibody is in the tumor, cleavage by a resident proteasereleases the EGFR domain III epitope, allowing the antibody to bind itstarget. FIG. 8B is a schematic view of the overall design to generateIgGs that are masked and do not bind antigen in normal tissues.Cross-masking permits the simultaneous delivery of two antibodies thatsynergize or target independent oncogenic signaling pathways.

In another embodiment of the disclosure, masked anti-EGFR antibodyfragments are generated by cloning domain III of the soluble EGFR(sEGFRdIII) N-terminal to a cleavable linker followed by single chainvariable fragment (scFv) versions of the anti-EGFR antibodies Matuzumab(mAb425 or 425) and Cetuximab (mAbC225 or C225) (FIGS. 9A and 17). Insome embodiments, a metalloprotease 9 (MMP-9) substrate cleavage site,VPLSLYS (SEQ ID NO:3) (Turk et al., 2001) may be encoded within thecleavable linker. MMP-9 is frequently overexpressed in epithelialmalignancies in which EGFR blockade may have therapeutic benefit(Swinson et al., 2004; Cox et al., 2000; Zhou et al., 2006).

In another embodiment, a masked anti-HER2 antibody or fragment thereofis generated by cloning domain IV of the HER2 receptor to a cleavablelinker followed by an Fc fragment of the anti-HER2 antibody, Trastuzumab(FIG. 27). In some embodiments, a metalloprotease 9 (MMP-9) substratecleavage site, VPLSLYS (SEQ ID NO:3) (Turk et al., 2001) may be encodedwithin the cleavable linker.

In one embodiment, a linker that was significantly longer than theminimal possible distance required may be used to address potentialgeometric problems of epitope association with the scFv and taking intoconsideration that affinity decays slowly as a function of linker length(Krishnamurthy et al, 2007). In such an embodiment, the serine-glycinerich linker has 12 and 19 residues flanking an MMP-9 sequence, producingan end-to-end length of approximately 133 angstroms (Å). Crystalstructures have revealed the distance between the C-terminus ofsEGFRdIII and the N-terminus of the antibody light chains is >35.1 Å forC225 (Li et al., 2005) and 34.7 Å for 425 (Schmiedel et al., 2008).

In further embodiments, the scFv may be replaced by an IgG, whichcreates a bivalent format. In this case, overall affinity to thetumor-derived antigen may be significantly enhanced due to energyadditivity for the following reasons. First, FIG. 2B illustrates thatthe bivalent IgG 425 binds with greater affinity than the monovalentscFv. Second, cleaved masking epitopes (which are monomeric) competepoorly with tumor-derived EGFR for the unmasked antibody, and thebivalent interaction with cells may be favored over the monovalent maskinteractions. Thus, the cleaved masking epitope is free to diffuse fromthe tumor site, favoring a higher affinity for the antibody. Moreover,the inclusion of an antibody Fc region enables downregulation of EGFR(Friedman et al., 2005) as well as antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Yan etal., 2008). Third, assuming no new constraints, the occlusion of theantibody binding sites may improve with the use of IgGs, as the maskingwill be tetravalent. The gain in affinity in the tetravalent formatpermits the inclusion of additional mutations in the mask to furtherenhance the dissociation rate of the masks upon protease cleavage.

In other embodiments, a masked antibody may comprise a protein A-proteinL (protein A-L) mask that binds to a monoclonal antibody. Protein A(which binds the heavy chain of an antibody variable region) and ProteinL (which binds the light chain of an antibody variable region), are twoproteins known to bind antibodies, and in some embodiments, may beattached by a protease cleavable linker to take advantage of avidityproviding tighter binding than either protein alone (see Example 4below). Once the masked antibody reaches the tumor cell, the proteasesite will be cleaved by tumor over-expressing proteases, avidity will belost, and the mask will dissociate allowing the antibody to bind at thetumor site (FIG. 33). The mask will not be significantly cleaved inhealthy tissues and therefore will prevent off target binding. In oneembodiment (as described in detail in the Examples below), the proteinA-L mask binds Trastuzumab, but the protein A-L mask may be used to maskany suitable therapeutic antibody known in the art including, but notlimited to, alemtuzumab, bevacizumab, Cetuximab, edrecolomab,gemtuzumab, ibritumomab tiuxetan, Matuzumab, panitumumab, rituximab,tositumomab, and Trastuzumab.

Crystallization of some antibodies has traditionally been challengingdue to flexibility of the antibodies. One method of inducingcrystallization may include addition of a ligand that changes thesolubility of the protein. Therefore, in addition to forming a maskedantibody, Protein A, Protein L or a combination of both may be used as‘additives’ for Fabs that do not readily crystallize and/or to improvethe diffraction limits of the same.

The following examples are provided to better illustrate the embodimentsand are not to be interpreted as limiting the scope of any claimedembodiment. The extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention. Although the examples described below arefocused on EGFR and HER2 mAbs, one skilled in the art would understandthat the design strategy is broadly applicable to other mAbs and/orcombinations used to treat cancer and chronic inflammation. Further. allreferences cited above and below are incorporated by reference as iffully set forth herein.

EXAMPLE 1 EGFR Antagonistic mAbs C225 and 425 Cause Enhanced EGFRInhibition and Distinct Epitope Recognition

Materials and Methods

Cells and Reagents.

Human epithelial breast cancer cells MDA-MB-468 (ATCC HTB-132) werepurchased from American Type Culture Collection (ATCC), Rockville, Md.,USA. NIH3T3 cells transfected with either full-length human EGFR (CO12)or mutated EGFR variant III (EGFRvIII; HC2) were a generous gift fromDr. Albert Wong (Moscatello et al., 1996). MAb 425 was isolated frommurine ascites by affinity purification using Protein A Sepharosecolumns followed by Ion Exchange columns (GE Health Sciences Q Sepharose4, fast flow). Purified C225/Cetuximab was purchased from Bristol MyersSquibb, Princeton, N.J. The EGFR selective tyrosine kinase inhibitor,tyrphostin AG1478 (AG1478), was purchased from Calbiochem. For surfaceplasmon resonance experiments, the extracellular soluble domain of thehuman EGFR (sEGFR) was purchased from R&D Systems, Inc., WST-1 cellproliferation kit was purchased from Takara Bio Inc. HC2, CO12, andMDA-MB-468 cells were cultured in DMEM supplemented with 1 g/l glucose,2 mM L-glutamine, 10% FBS, 1% penicillin/streptomycin under standardconditions.

Expression of Recombinant EGFR Fragments.

Soluble EGFR (sEGFR) constructs (including, but not limited to, anysoluble EGFR construct including approximately residues 1-618 toresidues 1-629), extracellular domain (residues 1-621) and domain III,(residues 310-512) were generated, produced and purified closelyfollowing published methods (Ferguson, 2004). Two point mutations,S460P/G461N, were introduced into sEGFR domain III by site directedmutagenesis using the QuikChange method (Stratagene).

Papain Digestion of 425 and C225.

Fab fragments of 425 and C225/Cetuximab were prepared by papaindigestion and Protein A reverse purification (Pierce). Each protein wasfurther purified using a HiLoad 16/60 Superdex 75 column.

Flow Cytometry.

Flow cytometric analyses were carried out using mAbs 425 and C225conjugated to Alexa Fluor 488 through primary amines following themanufacturer's protocol (Molecular Probes). Between 4-6 Alexa 488molecules were bound per antibody as estimated by measuring the opticaldensity at 280 nm and 494 nm. For FACS analysis, cells were detachedusing a non-enzymatic cell dissociation solution (Cellgro), collectedand resuspended in wash buffer (1×PBS containing 1% BSA). Approximately500,000 cells were incubated at 4° C. in 50 μl of labeled and unlabelledantibodies as indicated. After 30 minutes of incubation, cells werewashed three times with wash buffer and fixed using 1% freshly preparedparaformaldehyde. Samples were analyzed on a FACS Canto (BDBiosciences).

Wst-1 Assay.

Effects of C225 and 425 on metabolism of MDA-MB-468 were measured byassaying cleavage of the tetrazolium salt WST-1 to fluorescent formazanby cellular mitochondrial dehydrogenases as quantified by measuring theabsorbance of the dye solution at 450 nm. In 48-well plates,approximately 3000 cells/well suspended in 200 μl of DMEM containing 10%FCS were allowed to attach for 24 hours. After 24 hours, 100 μl ofantibody solutions diluted in DMEM were added to each well to achievethe desired concentrations. After 72 hours, 30 μl of WST-1 was added foranother three hours. For analysis, 60 μl of culture medium was added to200 μl of 1×PBS buffer and the absorbance was measured at 450 nm using aVictor2 1420 Multilabel counter (Perkin Elmer). The absorbance at 550 nmwas used for background correction. The percent inhibition wascalculated as 100*(AbsControl−AbsmAb)/AbsControl.

Anchorage-Independent Cell Growth and Survival.

Cell survival in the anchorage-independent state was determined aspreviously described (Jost et al., 2001a) with minor modifications. Cellsuspensions were prepared in DMEM containing 0.2% BSA in the presenceand absence of 10% FCS, mAbs 425, C225 or their combination (10 μg/mlfinal antibody concentration), AG1478 (10 μM) and/or EGF (10 ng/ml).After 48 and 72 hours, 500 μl of cell suspension was transferred toanother E-well plate with the respective culture medium and allowed toattach and grow for 24 hours. Attached cells were fixed in 75% ethanoland stained with crystal violet.

Immunoblot Analyses.

Cells were incubated in complete growth medium in 100 mm petri dishes(1×10⁶ cells per dish) for 24 hours. After overnight incubation inserum-free DMEM containing 0.2% BSA, antibodies (10 μg/ml final IgGconcentration) or AG1478 (10 μM) were added. After one hour, EGF (10ng/ml final concentration) was added to culture media and cells lysedusing Laemmli buffer. Differences in the phosphorylation of MAPK, AKTand EGFR were determined by immunoblot analysis. Antibody binding wasdetected using an enhanced chemiluminescence system (Pierce).

Biacore Surface Plasmon Resonance Analysis.

Molecular interactions were determined using a Biacore® 3000 opticalbiosensor (Biacore Inc.,). Immobilization of EGFR specific mAbs to CM5sensor chips were performed following the standard amine couplingprocedure. Unless specified otherwise anti-HIV-1 gp120 antibody 17bimmobilized on CM5 chips was used as a reference flow cell (Thali etal., 1993). Ligand densities and flow rates were optimized to minimizemass transport and rebinding effects.

Analysis of direct binding of sEGFR in a concentration dependent mannerto 425 or C225 was achieved by passage over mAb surfaces with a liganddensity of 200 RUs and a flow rate of 50 μl/min for two minutesassociation and 6 minutes dissociation at 25° C. Regeneration of thesurfaces between injections was achieved by injecting three, six secpulses of 10 mM glycine, pH 2.0 at the flow rate of 100 μl/min.

To study simultaneous binding of 425 and C225, a capture SPR format wasemployed. Briefly, sEGFR (5 nM) was injected over a low density C225surface (280 RUs) at 20 μl/min. The captured sEGFR was then used asligand to perform saturation analysis by injecting increasingconcentrations of 425 (0-512 nM) for three minutes at a flow rate of 50μl/min until binding equilibrium (Req) was achieved. Data were analyzedusing BIAevaluation® 4.0 software (Biacore Inc., NJ). The responses of abuffer injection and responses from a reference flow cell weresubtracted to account for nonspecific binding and instrument noise.Experimental data were fitted to a simple 1:1 binding model with aparameter included for mass transport.

Sedimentation Equilibrium Analysis.

A complex of full-length sEGFR, Fab425 and FabC225 was incubated for 30minutes, applied to a HiLoad 16/60 Superdex 200 prep grade column andloaded into a 6-well, analytical centrifugation cell at A280 nm=1.0. Thesamples were centrifuged using an An-50 Ti rotor at 20° C. in a BeckmanProteomeLab XL-I ultracentrifuge. Absorbance scans at 280 nm wereperformed after 12 and 14 hours at 8000, 12000 and 16000 RPM.Equilibrium was assessed by comparison of scans at 12 and 14 hours.Analysis was performed using FastFitter (Arkin and Lear, 2001) asimplemented in Igor Pro (Wavemetrics, Lake Oswego, Oreg.). The solventdensity (ρ) was set at 1.0042 g/ml and the specific volume (VBAR) wasassumed to be 0.76 ml/g.

Size Exclusion Chromatography.

Complexes comprised of different combinations of sEGFR domain III, themutated (S460P/G461N) sEGFR domain III, Fab 425 and Fab C225 wereprepared at 4 μM and incubated for 20 minutes. Size exclusionchromatography was performed at 4° C. using a Superdex 200 HR10/30column (GE Health Sciences) and monitored at 280 nm

Cooperative Inhibition of Growth and Survival of MDA-MB-468 BreastCarcinoma Cells by mAbs 425 and C225.

Combinations of mAbs binding to different epitopes of the same antigenhave proven to exert synergistic effects against tumor cells expressingtheir cognate antigens at the cell surface. For example, two antibodiesto ErbB2 (i.e., Trastuzumab and Pertuzumab) inhibit survival of breastcancer cells more effectively than either antibody alone (Nahta et al.,2004).

The antibodies 425 and C225 both have the capacity to inhibit ligandbinding to the EGFR independently, but act synergistically (i.e., a muchlower dose of the antibody combination achieved the same biologicalresponse) to affect breast tumor cell growth and survival. The C225binding epitope is in direct competition with EGF/TGFα for binding todomain III of the extracellular portion of the EGFR (Gill et al., 1984;Li et al., 2005). MAb 425 interferes with ligand access to the EGFR(Murthy et al., 1987), but the binding site for 425 is currentlyunknown. The effects of either antibody alone were compared with thoseof the combination of both antibodies on growth and survival ofMDA-MB-468 breast carcinoma cells that express high levels of EGFR(Biscardi et al., 1998). To avoid effects due to differences in antibodyconcentration the total amount of IgG was kept constant for allexperimental conditions. As shown in FIG. 1A, the combination of the twoantibodies is superior to either antibody alone in inhibiting metabolicactivity of actively growing, attached MDA-MB-468 cells. The synergisticeffect of the antibody combination at 25% growth inhibition isdemonstrated by isobologram which depicts equally effective dose pairs(isoboles; FIG. 1B). In this representation, the concentration of onedrug required to produce a desired effect is plotted on the horizontalaxis while the concentration of another drug producing the same effectis plotted on the vertical axis. A straight line joining these twopoints represents additive effects expected by the combination of twodrugs. FIG. 1B shows that, at 25% growth inhibition, the experimentalvalue for the antibody combination lies well below the theoreticaladditive line consistent with drug synergism. This demonstrates that thecombination of the two antibodies is superior to either antibody alonein inhibiting metabolic activity of actively growing MDA-MB-468 cells.

The capacity of the antibody combination to induce cell death in theanchorage-independent state is illustrated in FIG. 1C. It has beenpreviously demonstrated that EGFR inhibition with either 425 (10 μg/ml)or with small molecule tyrosine kinase inhibitors accelerates apoptosisof epithelial cells maintained in forced suspension culture whichprecludes extracellular matrix attachment (Jost et al., 2001a; Jost etal., 2001b). A simple method to assay cell survival in these conditionsconsists of reseeding cells on tissue culture-treated plastic afterdefined periods of suspension culture and the determination of cellreattachment after 12-24 hours. Treatment with either antibody at 10μg/ml during suspension culture reduced the number of cells capable ofmatrix reattachment only marginally irrespective of the culture mediumused in these experiments (FIG. 1C). In contrast, the combination ofboth antibodies where each antibody was used at 5 μg/ml markedly reducedlevels of viable reattached cells similar to cultures treated with thesmall molecule EGFR inhibitor AG1478. This indicates that the antibodycombination accelerates death of MBA-MB-468 cells in theanchorage-independent state.

Inhibitory Effects of the C225/425 Antibody Combination on SignalTransduction Events Triggered by EGFR Activation.

To account for combinatorial inhibitory effects of the C225/425 antibodycombination on EGFR-dependent signal transduction events, the effects ofthe antibodies used either singly or in combination on short-termEGF-induced signal transduction events were determined in serum-starvedMDA-MB-468 cells. This revealed more efficient inhibition of AKT andp42/44MAPK phosphorylation in serum-starved cells exposed to EGF and theantibody combination as compared to single antibody treatment (FIG. 2).Moreover, in MDA-MB-468 cells, 425 treatment alone did not inhibit AKTand MAPK phosphorylation, whereas it effectively reduced EGF-dependentphosphorylation of the EGFR on Y1068. As in the case of cell growthinhibition experiments, the effects on signal transduction eventsoccurred although either antibody was used at half the concentration (5μg/ml) when combined as compared to single antibody treatments (10μg/ml). Therefore, the C225/425 antibody combination effects weresimilar to those achieved by using AG1478 at very high concentration (10μM).

Simultaneous Binding of 425 and C225 to the Extracellular Domain of theEGFR.

Cooperative growth inhibitory effects by the two antibodies can beexplained by the binding of these antibodies to distinct EGFRpopulations thus providing more effective ligand binding competition.Alternatively, the two antibodies may simultaneously engage distinctepitopes of the EGFR domain III and inhibit EGFR-dependent signaltransduction by independent mechanisms. To distinguish between these twopossibilities, surface plasmon resonance experiments were performedwhereby either antibody was immobilized on a CM5 chip and successivebinding of soluble extracellular domain of the EGFR and the secondantibody was monitored. First, binding of sEGFR to either 425 or C225immobilized on the chip was characterized, as shown in FIGS. 3A and 3B.Lower RUs (˜200 RU) of mAbs were conjugated to avoid mass transportlimitations. Additionally, sEGFR was injected at a high flow rate of 50μl/min in order to overcome potential receptor rebinding effects. Theresultant sensorgrams were then analyzed and the equilibrium rateconstants were calculated. Each different concentration of injectedsEGFR is represented by a real time sensorgrams (lighter, wavy lines)while the calculated kinetic fit of each interaction is represented bysuperimposed smooth black line. The results showed that C225 binds tosEGFR with a higher affinity (2.7±0.4 nM) compared to 425 (32.3±6.75 nM)due to a comparatively higher dissociation rate of 425.

Next, it was determined whether sEGFR bound to C225 immobilized on thechip was capable of capturing 425. To this end, sEGFR (5 nM) wasinjected over a low density C225 chip (280 RU) followed by differentconcentrations of 425 (0-512 nM) until binding equilibrium was reached.The sensorgrams show binding of concentration-dependent binding of 425to sEGFR captured by C225 (FIG. 3C), consistent with noncompetitivebinding of the two antibodies to sEGFR.

To obtain independent confirmation for simultaneous binding of bothantibodies to EGFR, a sedimentation equilibrium analysis was performedby analytical ultracentrifugation using an admixture of C225, 425 andthe extracellular portion of the EGFR consisting of domains I to IV(sEGFR). This indicated a single species with an apparent weight of 167kD, consistent with the existence of a 1:1:1 tripartite molecularcomplex (FIGS. 4A-B). Note that total concentration was at 4.5 μMor >100-fold and >1000-fold the dissociation constant of 425 or C225 andEFGR, respectively. Together, these results strongly suggest that thebinding epitopes of C225 and 425 are distinct albeit both are confinedto domain III of the extracellular portion of the human EGFR (Lax etal., 1991).

Next it was determined whether both antibodies could also simultaneouslyengage the EGFR expressed on cell surfaces. NIH3T3 cells stablytransfected with either full length wild-type human EGFR (CO12 cells) ora mutated EGFR characterized by intragenic deletion of most of domain IIof the EGFR and prominently expressed in neoplasia (HC2 cells) were usedfor this purpose (Moscatello et al., 1996). Both antibodies bindexclusively to domain III of the EGFR (Lax et al., 1991), and thereforemay bind to both wild-type and tumor-specific EGFRvIII. To avoidconfounding effects of endogenous EGFR expression, transfected mouse 3T3cells were used rather than human cells. Since neither antibodyrecognizes the murine EGFR (Murthy et al., 1987; Wen et al., 2001), nobinding other than to the transfected human EGFR was measured. To assessdirect binding competition between the two antibodies, the ability of425 to replace C225 from the cell surface of CO12 and HC2 cells wasdetermined by FACS analysis. The results showed that each antibodycompetes with itself for cell surface binding but not with the otherantibody (FIGS. 5A-D). In addition, both antibodies recognized bothhuman wild-type EGFR and EGFRvIII. These results indicate that both C225and 425 antibodies independently and simultaneously bind to distinctepitopes on domain III of the extracellular portion of recombinant humanEGFR and cell associated EGFR.

C225 directly competes with ligand binding to domain III of the EGFR (Liet al., 2005) and 425 is also known to bind to domain III even thoughits epitope has yet to be defined. Therefore, the 425 binding site mayencompass residues that are different in the human and murine EGFRsequences, since 425 recognizes a conformationally defined epitope ofthe human but not the murine EGFR (Murthy et al., 1987). Residues thatdiffer between the murine and human sequences and are present on thesurface of the sEGFR domain III are likely candidates for the epitopedefined by 425 binding. In addition, because C225 and 425 bindsimultaneously to the surface of EGFR domain III, the 425 epitope likelylies outside of the surface masked by C225.

Mapping of the human EGFR using these constraints produced a handful ofpotential EGFR/425 interaction sites (FIG. 6). Many of these sites arelocated near glycosylation sites and were considered unlikely targetsfor 425 binding because it was previously shown that 425 recognizes aprotein epitope on the deglycosylated EGFR (Murthy et al., 1987). Afterexclusion of residues occluded by either C225 or by putativecarbohydrate side chains, two adjacent amino acids emerged as likelycandidates for 425 docking (Ser460 and Gly461 highlighted in FIG. 6). Toinvestigate the role of these residues in 425 binding, these tworesidues were changed in human EGFR domain III to the correspondingmurine sequence, (i.e., Pro460 and Asn461), expressed and purified thisdomain, and used size exclusion chromatography to test whether 425 couldbind sEGFR domain III encoding S460P and G461N mutations (FIG. 7). Theindividual Fabs and sEGFR domain III proteins eluted at 15.6 mL.Co-incubation of sEGFR domain IIIS460P/G461N with Fab425 resulted in aslightly earlier elution, 15.2 mL, indicating weak association. Theconcentration of the mixture added to the column is 4 μM, which isgreater than the KD of the native EGFR/425 interaction by a factor of125, indicating a weak association. Similarly, mutations that define theC225 epitope on EGFR-domain III reduce the affinity from 2.3 nM to 340nM (Li et al., 2005) and a complex formed by such a mutant and C225 alsoshow residual binding wherein the concentration is greater than theoriginal KD (e.g., ˜290 nM) by a factor of 125. However, human sEGFRdomain III at the same concentration forms a saturated complex thateluted at 13.9 mL. To demonstrate that the point mutations did notaffect the tertiary structure of the sEFGR domain III, the S460P andG461N mutant were also mixed with C225 Fab and subjected to analysis bysize exclusion chromatography. The resulting elution point at 14.0 mLwas similar to the wild-type sEGFR domain III complexed with C225.Finally, when wild-type sEGFR domain III and both Fabs were mixed andapplied to the column, a distinct peak eluting at 13.1 mL emerged,consistent with a tripartite complex. These data indicate that Ser460and Gly461 significantly contribute to the overall affinity of theEGFR-425 interaction.

Because 425 does not compete with C225 for binding and recognizessurface residues distinct from the ligand binding site, a differentmechanism of action that is independent of C225 and the ligand mayaccount for its biological effects. Specifically, interaction of 425with the EGFR may interfere with high affinity ligand binding byblocking a conformational change to bring domains I and III of theextracellular domain of the EGFR in close proximity.

EXAMPLE 2 EGFR-Specific scFvs Engineered to Enable Selective AntigenRecognition Upon Proteolytic Activation

Generation of C225 and 425 Masked mAbs

The masked scFvs were produced as secreted proteins from insect cellsinfected with baculovirus and were purified by Ni-affinity and sizeexclusion chromatography.

Domain III of the soluble epidermal growth factor receptor (sEGFRdIII,comprising residues 310-512) was cloned into pAcSG2 behind the humanEGFR secretion signal sequence. To this was added a flexible,glycine-serine linker containing the matrix metalloproteinase 9 (MMP-9)consensus protease site-VPLSLYS (SEQ ID NO:3) and finally a single chainvariable region (scFv) anti-EGFR antibody. The full linker sequence is(GGGSGGGSGGGSVPLSLYSGSTSGSGKSSEGSGSGAQG) (SEQ ID NO:4; MMP-9 proteasesite is underlined). Both scFv constructs of C225 and 425 were assembledV_(L)-linker-V_(H). C-terminal FLAG (425 only) and hexahistadine tags(C225 and 425) were also added. This vector was mixed with linearizedbaculovirus DNA (BD Biosciences), transfected and expanded for threerounds before confirming viral titers. Large-scale expression (5 L) wasconducted in suspension culture. The media was separated from cellularmaterial and diafiltered against purification buffer as describedpreviously (Ferguson et al., 2000). Protein purification wasaccomplished using a nickel-NTA column followed by a HiLoad 26/60Superdex 200 preparative column (Amersham).

Secreted masked mAbC225 scFv contained a mixture of the expected lengthand digested fragments. Analytical size exclusion chromatography (SEC)of the purified material indicated dimeric species, but no monomeric oroligomeric species. Treatment of the purified material with MMP-9 andanalysis by SEC indicated that the homodimeric complex was cleaved (FIG.13). In addition, SDS-PAGE of the protease-treated, homodimeric complexindicated that MMP-9 cleavage was specific, producing two bands of thepredicted molecular weight and no cleavage of the individual domains orscFv linker. Together, this suggests that the linker may interfere withthe intramolecular association of the epitope to the scFv (e.g., asingle masked species), potentially reflecting a combination of stericclashes and an entropic penalty. These data provide evidence that it ispossible to mask the complement-determining region (CDR) and cleave themasking agent.

Since a dimeric interaction was observed, a heterodimeric orcrossed-masked design was also produced to simultaneously deliver twotherapeutic antibodies to the tumor site was also considered. Theutility of such a design is justified by studies, as described inExample 1, which show that the combination of C225 and 425 actsynergistically to inhibit EGF-stimulated cell growth and survival(Kamat et al., 2008), and is more effective in elicitingcomplement-dependent tumor cell lysis than other combinations ofanti-EGFR antibodies with C225 (Dechant et al., 2008). To favor assemblyof ‘cross-masked’ 425/C225 complexes wherein the mask linked to C225scFv binds to 425 scFv (and vice versa), the native epitopes werealtered by introducing point mutations that diminish intramolecularaffinity but not intermolecular complex formation. Hexahistidine andFLAG tags facilitated purification and detection. Purification byaffinity and size exclusion chromatography of the masked 425 and C225scFvs yielded undigested material of greater than 95% purity (FIG. 9C).Despite the multivalent nature of the constructs, size exclusionchromatography did not reveal the presence of aggregates.

Purified masked 425 [sEGFRdIII (S460P/G461N)-scFv425; FIG. 15; SEQ IDNO:2 (nucleic acid); SEQ ID NO:6 (amino acid)] and masked C225[sEGFRdIII (Q384A/Q408M/H409E)-scFv C225; FIG. 14; SEQ ID NO:1 (nucleicacid); SEQ ID NO:5 (amino acid)] were allowed to associate for 20minutes at 4° C. and then loaded onto a Superdex 200 10/300 GL.Fractions corresponding to the crossmasked reagent were concentratedusing a Centricon Spin Concentrator (10 kDa, Millipore). This materialwas then exchanged 4 times into 2 volumes of reaction buffer: 50 mMTris, pH 7.4, 150 mM NaCl, 5 mM CaCl₂, 0.02% Nonidet P-40 substitute(Backstrom et al., 1996). Active, recombinant MMP-9 (Calbiochem) wasincubated with the cross-masked 425/C225 and masked scFvs at a molarratio of 1:42. The completeness of the reaction was assessed by reducingSDS-PAGE and was at least 95% complete by Coomassie staining. The masked425 and masked C225 scFvs formed a cross-masked, non-covalent 425/C225scFv complex, which eluted from a size exclusion column as a symmetricpeak consistent with the calculated molecular mass of 106 kDa (FIG. 9B).Both antibody derivatives were completely cleaved by recombinant activeMMP-9 into the mask and scFv proteins (FIG. 9C). Samples wereimmediately frozen at −20° C. until needed for experiments.

Affinity of C225 and 425 Masked mAbs for EGFR Domain III.

Surface Plasmon Resonance Interaction Studies.

The affinity of masked antibodies for immobilized sEGFRdIII wasdetermined using surface plasmon resonance (SPR) analysis. F(ab)′fragments of the parental antibodies mAb425 and mAbC225 were used ascontrols to verify the immobilization and stability of the sEGFRdIIIover multiple analytical cycles (see supporting data). The affinity ofF(ab)′ 425 and C225 were 91±23 and 5.3±0.6 nM, respectively (Table 1,below). The scFv constructs bound with weaker affinity of 260±40 and110±20 nM, respectively. After characterizing these antibody fragments,the masked antibodies were measured singly or as a heterodimer beforeand after MMP-9 digestion. For masked C225 scFv and masked 425 scFv, nodifference in affinity as compared to the respective unmasked scFvs wasobserved, demonstrating that the point mutations interfere withself-association. However, the CDRs of the cross-masked 425/C225 scFvswere effectively occluded, as shown by weak binding to sEGFRdIII. Incontrast, treatment of cross-masked 425/C225 with MMP-9 increasedbinding affinity by approximately an order of magnitude. Representativetraces are shown at 1 μM and 100 nM in FIGS. 10A and 12A-B.Specifically, in the absence of MMP-9, the affinity for sEGFRdIII was3.5±1.1 μM, but after MMP-9 exposure the affinity increased to 420±270nM

Binding experiments were performed on a BIAcore T100 instrument at 25°C. in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 8.0 and0.005% Tween 20). Immobilization was accomplished using standard aminecoupling to a CM5 chip. Blank immoblization was used for the referencecell. sEGFR domain III was applied at 50 μg/mL in acetate buffer, pH 5.5for a target of 5000 response units (3310 RU final). Bioactivity of thechip was confirmed by steady state binding of Fab225 at 30 μL/min. Allother analyses were conducted at the same flow rate. Binding wasassessed at 1 μM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM. Theassociation phase was 380 seconds and dissociation 300 seconds.Regeneration was accomplished using 30 μL of 100 mM glycine, pH 3.0 at aflow rate of 90 μL/min. Steady state measurements were fit to theexpression RU=(R_(max)*[ ])/([ ]+K_(d)). Each dissociation constant wasdetermined at least three times. The bioactivity of the chip graduallydeclined over time as a result of the regeneration conditions. However,minimal variation within a run permitted analysis.

TABLE 1 Dissociation constants (K_(D)) of antibody fragmentsDissociation Constant* (nM) Antibody Fragments (−) MMP-9 (+) MMP-9 RatioCross-masked 3500 ± 1100 420 ± 270 8.26 ± 5.92 425/C225 Masked 425 240 ±100 370 ± 110 0.66 ± 0.34 Masked C225 560 ± 800 570 ± 770 0.99 ± 1.93scFv 425 260 ± 40  2.87 ± 0.83 Fab 425 91 ± 23 scFv C225 110 ± 20  21.5± 4.7  Fab C225 5.3 ± 0.6 *n = 3

Flow Cytometry Interaction Studies.

To measure differential affinity in the context of EGFR expressed ontarget cell surfaces, cross-masked 425/C225 antibody binding tokeratinocyte-derived HaCaT cells that express EGFR was also tested.Binding was detected by flow cytometry using an AlexaFluor-labeledanti-FLAG antibody. The antibody only detects the masked 425 becausemasked C225 lacks a FLAG tag. First, the association of masked 425 withHaCaT cells showed little dependence on MMP-9 digestion (FIG. 13). Thisis consistent with the intramolecular mask being unable to occlude thelinked 425 antibody and consistent with the SPR results. Next, it wasobserved that the cross-masked 425/C225 had low affinity for the HaCaTcells (FIG. 10B), whereas cleavage of the antibody derivatives withMMP-9 markedly enhanced their binding (FIG. 10B). Binding of thedigested cross-masked 425/C225 scFvs was comparable to that of the 425scFv alone (FIG. 10B), indicating that the N-terminal extensionremaining after digestion does not compromise binding.

Flow cytometric analyses were carried out using mAb425 and anti-FLAG(clone M2, Sigma) amine-conjugated to Alexa Fluor 488 according to themanufacturer's protocol (Molecular Probes). Between 4-8 Alexa 488molecules were bound per antibody as estimated by measuring the opticaldensity at 280 nm and 494 nm. For FACS analysis, HaCaT cells weredetached with trypsin/EDTA. The trypsin was inactivated with DMEM/FBSand the cells were collected and resuspended in wash buffer (1×PBScontaining 1% BSA). Approximately 1,000,000 cells were incubated at 4°C. in 50 μl of digested and undigested cross-masked 425/C225 asindicated. After 45 minutes of incubation, cells were washed twice withwash buffer and incubated for 20 minutes with the anti-FLAG secondaryantibody or mAb425. Samples were analyzed on a FACSCalibur (BDBiosciences). The specificity of scFv 425 was confirmed bypre-incubation with unlabeled mAb425. scFv C225 did not yield any signalabove the anti-FLAG control since it lacks the FLAG tag (FIG. 13).

Expression and Cleavage by MMP9 of Cetuximab Masked with EGFR Domain IIILinked Via a Cleavable or Non-Cleavable Linker.

Mask Design.

FIG. 18 shows the Design of a mask. Briefly, EGFR domain III is fused toheavy chain of Cetuximab via a MMP9 cleavable (top line) ornon-cleavable linker (bottom line). The mask-heavy chain and light chainof Cetuximab are cloned into a dual expression GS selection vector.

Transient Transfection.

4 μg of dual vector and 10 μl of Lipofectamine 2000 were mixed in 500 μlof Opti-MEM and applied to 0.5×10⁶ COS 7 cells in 6-well plates. Culturesupernatants were collected 72 h post-transfection. FIG. 19 (left) showstransient and stable expression of mask Cetuximab by Western blot withan anti-human Fc-HRP antibody of COS-7 culture supernatants three daysafter transient transfection.

Stable Cell Line Transfection and Selection.

100 μg of linearized dual vector were nucleofacted into 5×106 NS0 cellsand plated into 96 wells plates by limited dilution 24 h later. Singlecolonies were assayed for expression 3 weeks later, and positivecolonies were expanded. The right panel of FIG. 19 shows dot blotidentification of stably-transfected mask-Cetuximab NS0 cell lines.Culture supernatant of single colony clones were spotted and blotted byanti-human Fc-HRP antibody. This indicates that masked Cetuximab can bestably expressed in NS0 cells.

MMP9 Cleavage.

Substrate proteins were exchanged into 50 μl MMP9 cleavage buffer. 40-80μg of MMP9 were added and incubate at 37° C. for at least 3 h toovernight. FIG. 20 shows a non-reducing (left) and reducing (right)SDS-PAGE of mask Cetuximab with cleavable (WMC) and non-cleavable (XMC)linker, confirming presence of indicate proteins. FIG. 21 establishesthat the masked Cetuximab with the cleavable linker is able to becleaved by MMP9 in vitro.

Size Exchange Chromatography.

80 μl of protein in PBS were separated on a Sephrodex 200 analytical SECand were eluted into PBS in 0.5 ml fractions at 4° C. Both WMC and XMCcould form a complex with Matuzumab, indicated by the shift in theelution peak. Peak fractions from XMC+M425, XMC and M425 were resolvedon a non-reducing SDS-PAGE. Mask Cetuximab did not form a complex withCetuximab (FIGS. 23 and 24).

Ultracentrifugation (AUC).

100 μl of 1 mg/ml of isolated complexes were ultra-centrifuged in PBS at3000, 6000, 9000, 12,000, 15,000 rpm at 20° C. FIGS. 22A-E show thedetermination of the molecular weight of the referenced samples and thestoichiometry of the mask Cetuximab-Matuzumab cross-masked complex.Masked Cetuximab was shown to be able to form a complex with Matuzumabin a 1:1 ratio.

FACS.

Alexa 647-labeled WMC/XMC complexed with M425 were isolated from SEC, 50μl of 5 nM complexes treated with MMP9, and mixed with 0.5×106trypsinized MDA-MB-468 cells in 50 μl of 1% BSA in PBS. M425 werelabeled with Alexa 488 anti-human IgG secondary antibodies. MMP9cleavage released masked Cetuximab-matuzamb complex for binding to EGFRover-expressing tumor cells (FIGS. 25 and 26).

EXAMPLE 3 Non-Covalent Masking of Trastuzumab with a HER2 DomainIV-MMP9-Fc Bivalent Mask

Construct Design.

HER2 domain and the Fc portion of an IgG1 were connected by a flexiblelinker containing a MMP9 cleavage site (top sequence underlined). As acontrol, a version with a mutated MMP9 cleavage site (bottom sequenceunderlined) was also constructed (FIG. 27).

Expression and Purification.

The constructed were separately cloned into a pAcGP67A baculovirustransfer vector. Each vector was co-transfected with BD BaculoGoldLinearized Baculovirus DNA into Sf9 cells for baculovirus production.After three rounds of amplification a virus titer of about 2×10⁸/mlvirus particles was reached. High Five cells at 2×10⁶/ml culture densitywere transfected with the baculoviruses at MOI of 3. The culturesupernatants were harvested 72 hrs post transfection. The retrievedmedia were clarified and passed through a Protein A agarose bead column.The column was washed extensively with PBS, and the H4Fc was eluted with0.1M glycine at pH3 into 0.1 volume of 1M Tris at pH9 forneutralization. FIG. 28 shows that the transfection and culture producedthe H4Fc dimer, which is 79 kDa. The band below the dimer is likely theFc alone, indicating that some degradation occurred during expression.The also protein tends to aggregate at high concentrations. Therefore,the eluted H4Fc was further purified on a Superdex 200 size exclusioncolumn. The protein was eluted from about 170-195 ml, or fractions 22 to24. A portion of the SEC trace and a non-reducing SDS-PAGE (FIG. 29)show the H4Fc eluted from fractions 22-24, as well as two aggregationspecies in F17 and F20 and a degradation product, likely the Fc portion,in F26.

Binding of H4Fc to Trastuzumab. The purified H4Fc with either thewildtype or the mutated MMP9 cleavage sequence were tested by SPR forbinding to the Trastuzumab. Fab portion of Trastuzumab was immobilizedon the CM5 chip, and the H4Fc's were flown over the chip (FIG. 30). Forthe wildtype H4Fc, ka=1.377E+4 1/Ms, kd=8.641E-4 1/s; KD=6.277E-8 M; asfor the mutated version, KD=8.979×10^−8 M; ka=1.344×10^4, kd=0.001207.

MMP9 Cleavage.

The presence of the MMP9 cleavage within linker is to release the maskfrom Trastuzumab when the complex reaches the tumor microenvironment. Totest if the MMP9 linker is active when the mask is in complex withTrastuzumab, the H4Fc's were incubated with Trastuzumab at equal molarconcentration for 20 min, room temperature, and then MMP9 was added foran overnight incubation at 37° C. The proteins were resolved on anon-reducing SDS-PAGE (FIG. 31). As expected, the H4Fc with the wildtypeMMP9 cleavage site was cleaved in the presence or absence ofTrastuzumab, while the mutated H4Fc showed no cleavage by recombinantMMP9.

H4Fc Inhibits Trastuzumab Binding to SKBR3 Cells.

Alexa 488 labeled Trastuzumab was incubated with either H4Fc's at a 1:1or 1:2 ratio for 20 min at room temperature, and then MMP9 was added forincubated an overnight incubation at 37° C. The mixtures were thenapplied to trypsinized SKBR3 cell suspensions for 1 hr, washedthoroughly, and subjected to FACS analysis. As shown in FIG. 32, thebinding of the mAb to SKBR3 cells showed a dose-dependent inhibition bythe increasing amount of H4Fc in the system. The addition of MMP9 to thewildtype H4Fc containing sample restored some binding of the mAb tocells. On the other hand, the mutated H4Fc containing samples showed nodifference whether MMP9 was added or not.

EXAMPLE 4 Modulation of Antibody Binding Kinetics Via Non-CovalentMasking

Diffraction Studies of Protein A and Protein L Bound to Trastuzumab.

The Trastuzumab Fab was co-crystallized with protein A and protein L.The crystal structure provides the basis to rationally design stericmasks activated at the tumor microenvironment. As determined by thestructures (FIGS. 34A-D) and the X-ray diffraction data for theTrastuzumab Fab (FIG. 35), Protein A and L have been shown to be usefulas ‘additives’ for Fabs that do not readily crystallize and/or toimprove the diffraction limits. The data set was collected at SSRL, thestructure was solved by MR and refined using phenix software. Maps werecontoured at 1.0 and figures were made using pymol.

Hydrodynamic Analysis.

To characterize the interaction between the protein A-protein L mask andthe Trastuzumab Fab, a sedimentation equilibrium analysis was performedby analytical ultracentrifugation, using an admixture of proteinA-protein L mask (FIG. 36B), Trastuzumab Fab (FIG. 36C), and the proteinA-protein L mask and Trastuzumab Fab (FIG. 36D). As shown by FIGS. 36A-Dand the data in Table 2 (below), the hydrodynamic studies indicate a 1:1complex.

TABLE 2 Expected and calculated masses derived from sed. eEq.experiments Sample Expected MW AUC Experimental MW mask 14,985 16,082+/− 162.70 Fab 46,755 45,909 +/− 166.41 mask + Fab 61,740 56,380 +/−160.33

SPR Analysis.

FIGS. 37A-C show that the protein L-A mask binds with higher affinitythan protein A or protein L alone as illustrated by surface plasmonresonance (SPR) analysis of the binding affinity of protein L (FIG. 37a), protein L (FIG. 37b ) and protein A-protein L mask for TrastuzumabFab (FIG. 37c ), The concentration range for a-b is 3.2 nM to 5 μM and10 pM to 1 μM. The binding affinity values are summarized in Table 3(below).

TABLE 3 Sample KD chi2 Protein A 29.98 uM 0.0157 Protein L 173.5 nM 17.5Protein A-Protein L mask 86.2 nM 2.04

FIGS. 38A-D show a comparison of on-rates of masked and unmaskedTrastuzumab Fab binding to HER2 by illustrating an SPR Analysis ofTrastuzumab Fab binding to HER2 (FIGS. 38a-38b ) and Trastuzumab Fabwith excess protein A-protein L mask binding to HER2 (FIGS. 38c-38d ).Panels b and d compare the on rates of the two Fab to HER2. The on-rateis slower for the masked, illustrating that an antibody can beeffectively masked to prevent off-target effects using a generic proteinA-protein L mask.

REFERENCES

The References listed below, and all references cited in thespecification are hereby incorporated by reference in their entirety asif fully set forth herein.

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What is claimed is:
 1. A cross-masked monoclonal antibody (mAb) complexcomprising: (1) a first masked mAb comprising: (a) a first maskingepitope comprising amino acid residues 23-198 of SEQ ID NO:5 as shown inFIG. 14; (b) a first linker that is cleavable by a protease specific toa target tissue, wherein the protease is matrix metalloproteinase-9(MMP-9); and (c) a first antibody or a first antigen-binding fragmentthereof that binds EGFR, wherein the antibody is cetuximab, and (2) asecond masked mAb comprising: (a) a second masking epitope comprisingamino acid residues 23-198 of SEQ ID NO:6 as shown in FIG. 15; (b) asecond linker that is cleavable by the protease specific to the targettissue, wherein the protease is MMP-9; and (c) a second antibody or asecond antigen-binding fragment thereof that binds EGFR, wherein theantibody is matuzumab.
 2. The cross-masked mAb complex of claim 1,wherein the first masked mAb and the second masked mAb form a dimer byocclusion of the first and second antigen recognition sites by thesecond and first masking epitopes, respectively.
 3. The cross-masked mAbcomplex of claim 1, wherein the first linker and the second linkercomprise the amino acid sequence VPLSLYS (SEQ ID NO:3).
 4. Thecross-masked mAb complex of claim 3, wherein the first linker and thesecond linker comprise the amino acid sequence SEQ ID NO: 4 or SEQ IDNO:
 7. 5. The cross-masked mAb complex of claim 1, wherein the firstmasked mAb comprises a first signal sequence and the second masked mAbcomprises a second signal sequence.
 6. The cross-masked mAb complex ofclaim 5, wherein the first masked mAb comprises the amino acid sequenceof SEQ ID NO:5.
 7. The cross-masked mAb complex of claim 5, wherein thefirst masked mAb is encoded by the nucleic acid sequence of SEQ ID NO:1.8. The cross-masked mAb complex of claim 5, wherein the second maskedmAb comprises the amino acid sequence of SEQ ID NO:6, wherein the aminoacid sequence of SEQ ID NO: 6 comprises amino acid P at position 175 andN at position 176.